Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutat hione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these fou r enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The pr otein amounts of wild-type and Cys85-->Ser enzymes purified by the latter g el were three to seven-fold greater than those of the other two enzymes pur ified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both puri fication methods yielded highly pure enzymes, while there were minor amount s of inter- and intra-disulfide forms in the IMAC purified enzymes except f or the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purif ied enzymes shifted all of their mass spectra of matrix-assisted laser deso rption/ionization-time of flight mass spectrometry toward low molecular-mas s regions, while addition of GSH to IMAC purified enzymes shifted the spect ra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cy s138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity p urification. This result was confirmed by isoelectric focusing. These findi ngs suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins. (C) 1999 Elsevier Science B.V. All rights reserved.